Chemically Induced Extracellular Ice Nucleation Reduces Intracellular Ice Formation Enabling 2D and 3D Cellular Cryopreservation

3D cell assemblies such as spheroids reproduce the in vivo state more accurately than traditional 2D cell monolayers and are emerging as tools to reduce or replace animal testing. Current cryopreservation methods are not optimized for complex cell models, hence they are not easily banked and not as widely used as 2D models. Here we use soluble ice nucleating polysaccharides to nucleate extracellular ice and dramatically improve spheroid cryopreservation outcomes. This protects the cells beyond using DMSO alone, and with the major advantage that the nucleators function extracellularly and hence do not need to permeate the 3D cell models. Critical comparison of suspension, 2D and 3D cryopreservation outcomes demonstrated that warm-temperature ice nucleation reduces the formation of (fatal) intracellular ice, and in the case of 2/3D models this reduces propagation of ice between adjacent cells. This demonstrates that extracellular chemical nucleators could revolutionize the banking and deployment of advanced cell models.


S3
For suspension cryopreservation, cells were seeded into U-bottom 96 well plates at the same density as the immediate pre-freeze counts for monolayer cryopreservation (typically 4.010 4 cells in 25 µL for all cell types). Solutions of 2 cryoprotectant were prepared, consisting of either 20 % DMSO (-IN) or 20 % DMSO in 100 % (v/v) PWW (+IN), and 25 µL was added to each well (total well volume 50 µL).
Final concentrations per well were 10 % DMSO or 10 % DMSO in 50 % PWW. Plates were placed in a -80 C freezer and allowed to cool at an uncontrolled rate, then stored for 24 hours at -80 C. The initial cooling rate generated by placing plates directly into the -80 C freezer is 9 °C/min, as established in Murray et al. 1 To thaw, all plates were removed from the freezer and 100 µL of warm (37 C) media was added to each well. Plates were placed in an incubator at 37 C for 10 minutes to ensure complete thawing. For monolayer cells, media was exchanged for fresh media then plates were incubated for 24 hours.
Suspension plates were centrifuged at 730 g for 5 minutes to pellet cells. Media was replaced with fresh media, then the total well contents was transferred to a flat-bottom 96 well plate to facilitate adherent culture. Cells were cultured for 24 hours post-thaw before viability assessment by resazurin reduction assay.

A549 And HepG2 spheroid formation in low-attachment U-bottom 96-well plates
Cells were seeded at either 4000 or 8000 cells per well in the inner 60 wells of a low attachment Ubottom 96 well plate (Corning, CLS7007). Plates were centrifuged at 2000 rpm for 5 min then placed in an incubator (37 C, 5% CO2) for 5 days before cryopreservation to facilitate spheroid formation.
Spheroids were monitored daily and spheroid diameter was measured from light microscopy images using imageJ, version 1.49.

Cryopreservation of spheroids in low-attachment 96 well plates
Spheroids were frozen in 50 µL of cryoprotectant (CPA) containing 10 % DMSO (-IN) or 10 % DMSO in 50 % (v/v) PWW (+IN), in U-bottom 96-well plates (Corning, NY). Plates were placed on a CoolCell® MP plate (BioCision, LLC, Larkspur, CA) and transferred to a -80 ℃ freezer, to allow a cooling rate of 1°C/min. After 24 h at -80 ℃, the frozen plates were removed from the freezer and 100 µL of warm media was added to each well for rapid thawing. Plates were incubated at 37 ℃ for 3 min, then centrifuged and media exchanged for fresh media. Thawed plates were incubated for 24 hours post-thaw prior to analysis.

Evaluation of spheroid viability and morphology post-thaw
After spheroids cryopreservation and thawing, a CellTiter-Glo® 3D Cell Viability Assay (Promega, G9682, USA) was used to quantify ATP present in spheroids 24 h post-thaw. Non-frozen spheroids of the same size were initially analysed and served as a control group. Each frozen (24 h) and thawed (24 h) spheroid was transferred to individual wells of a white 96 well-plate, the assay was performed and the resulting luminescence was measured in a plate reader.
The LIVE/DEAD® Viability/Cytotoxicity Assay Kit was used to observe apoptotic cell death and morphology of spheroids. Samples (8000 cells/spheroid/well) were washed twice with a DPBS buffer and treated with 200 µL solution/well containing 2 µM of calcein AM (5 µL) and 4 µM of ethidium homodimer-1 (20 µL) in sterile DPBS for 1 hour and then transferred to confocal dishes (VWR International Ltd., UK, 734-2904). Spheroids were imaged using a FV3000 confocal laser-scanning microscope (Olympus, Tokyo, Japan). The polyanionic dye calcein AM retained within live cells, showing an intense uniform green florescence in live cells at ex/em ~495 nm/ ~515 nm. EthD-1 entered cells with damaged membranes, binding to nucleic acids and producing a bright red fluorescence in dead cells at ex/em ~495 nm/ ~635 nm.

Reactive oxygen species (ROS) assay
Reactive Oxygen Species detection reagent (Invitrogen, D399) was both before and after-freezing spheroids (8000 cells/spheroid/well) imaging. Samples were washed twice with a DPBS buffer and incubated with 200 µL/well dichlorodihydrofluorescein diacetate (DCFDA) solution (20 µL of 7.5 mM diluted stock in PBS) for 30 minutes and then transferred to confocal dishes. The FV3000 confocal laser-scanning microscope (Olympus, Tokyo, Japan) was applied to image the spheroids with fluorescence excitation and emission in 492-495/505 and 517-527 nm.

Statistical analysis
Linear mixed effect (LME) models were used to test for the effect of ice nucleation (induced (+IN) vs passive (-IN)) on all three cell lines (A459, HepG2 and SW480) and cryopreservation formats (monolayer, suspension and spheroids). The response variables considered were normalised metabolic activity (nMA) in the monolayer and suspension formats, and normalised viability in the spheroid format. In both cases, nMA and viability are reported as percentages. In each case, several explanatory variables were considered and were coerced to factors prior to model fit. All LME models were fit using restricted maximum likelihood (REML) and were used to ensure that the variability associated with experimental replicates could be appropriately considered in the random effects distribution of the S5 model. In each case, stepwise model selection was conducted using ANOVA and Akaike information criterion (AIC). Model fit was assessed via visual inspection of the residuals. Models were fit separately to each cell line (A549, HepG2 and SW480) and to monolayer/suspension and spheroid, respectively.
All models were fitted in R (R Core Team, 2021; version 4.1.2) 2 using the lme4 package. 3 Data manipulation and visualisation made full use of the tidyverse collection of R packages 4 in particular the dplyr 5 and ggplot2 6 packages. Color palettes were sourced from the rcartocolor package. 7

Additional data Droplet Ice Nucleation Measurements
Ice nucleation temperatures of microlitre droplets were measured using a custom-built droplet freezing assay, as described previously. 1 The hornbeam pollen solution for nucleation measurements was prepared by adding 0.04 g Carpinus betulus pollen, purchased from Pharmallerga®, to 2 ml Milli-Q® water. The pollen suspension was refrigerated overnight before filtering through a 0.2 μm syringe filter into a clean glass vial. For the ice nucleation measurements, 40-50 microlitre droplets of the filtered, sterile pollen solution were pipetted, using a Sartorius Picus® electronic micropipette, on to a 22 mm diameter Hampton Research HR3-231 siliconized glass slide, placed on the aluminium plate of the cold stage. The cold stage temperature was lowered at a rate of 2 °C/min and the droplet freezing temperatures recorded. Nucleation temperatures of 1 μL Milli-Q® water droplets were measured using the same set-up for comparison. This demonstrates that the Hornbeam pollen used contains ice nucleators of the type used in our previous study on the use of pollen ice nucleators for cryopreservation. Figure S1 shows the ice nucleation data produced for this study and ice nucleation data for 1 µl droplets of Carpinus betulus PWW reported in Murray et al. 7 It can be seen that the Carpinus betulus PWW sample produced here has very similar ice nucleation properties to the sample used previously.
As such, we conclude that the larger volumes used for cryopreservation in 96-well plates will freeze at similar temperatures to those found in Murray et al. 7 using thermocouples embedded in 96-well plates. For comparison, Figure S1 also shows the data produced there for freezing of 100 µl volumes of PWW in 96-well plates.
Direct and accurate measurement of the ice nucleation temperatures in 96-well plates is challenging, and has only recently been accomplished using unique, bespoke infrared thermometry instruments. 11,12 By using 3 µl droplets we show that a larger fraction of droplets tends to freeze at warmer temperatures as increased volume of PWW is used, due to the presence of a rarer, more active ice nucleating macromolecules. In Murray et al. 7 it was estimated that Hornbeam PWW raises the nucleation temperature in 50 µl volumes of DMSO cryopreservation media to -8°C, high enough to expect substantially improved cryopreservation outcomes according to literature data. 4,5 We expect to see the same shift in nucleation temperature in this study. Future work should investigate the freezing temperatures of 50 µl volumes of PWW using infrared thermography. 11,12 Figure S1. Droplet fraction frozen for microlitre scale droplets of Carpinus betulus PWW (246 droplets) and Milli-Q® water (273 droplets). Also included is literature data from Murray et al. 7 for similar droplets, along with estimated freezing temperature ranges for pure water and Carpinus betulus PWW (shaded regions) produced using thermocouples embedded in 96-well plates to detect the latent heat of freezing.

Cryomicroscopy of A549 monolayer cryopreservation
The data in Figure 2B of the main text were produced by cryomicroscopy of the freezing of A549 cell monolayers grown on glass coverslips. The approach taken was inspired by work by Acker et al. 8 Cells were grown in the manner described in the 'cell culture' section above before being seeded onto circular glass coverslips placed on the bottom of 12-well plates, and allowed to adhere for 24 hours. To perform experiments, the cell culture media was removed and 20 µl of either 10% DMSO in cell culture media  Figure 2B to be determined. Only cells which were adhered to the surface were included. Figure S2 and Movie S1 show the cryomicroscopy process. Movie S1 shows both the IN-and IN+ conditions. While the observed changes in contrast are subtle, they are clear on video, and allow reasonably straightforward assessment of the proportion of cells darkening during cryopreservation. In two of the three experiments performed with control of ice nucleation no cells at all were observed to darken, while darkening was observed in many cells in the experiments where nucleation was not controlled. In all cases, there was a tendency for darkening of a cell to immediately precede darkening of neighbouring cells, in line with previous observations. 8 At the conclusion of each experiment the cells were rewarmed. In all cases the appearance of the cells was unchanged from the original pre-freezing state, although occasional non-adhered cells tended to move.

Statistical analysis by cell linemonolayer/suspension
A549. Induced ice nucleation was found to have a significantly positive effect on the normalised metabolic activity (nMA) of A549 cells post-thaw in the monolayer format (p-value < 0.001; Figure 2).
In comparison, no effect of induced ice nucleation on nMA was found in the suspension format (p-value = 0.29; Figure 2). Model selection via AIC demonstrates that the most parsimonious model includes a fixed effect interaction term between ice nucleation and cell format, as well as a random intercept term for experimental replicate (Table S2). This linear mixed effect model is found to explain 93% (R squared = 0.93; Table S2) of the variance in nMA. The model shows that both format and the variability associated with experimental replicate do have an effect on the relationship between ice nucleation and nMA. Model estimates show that the nMA is, on average, 26% higher in the monolayer format when ice nucleation is induced compared to passive (Table S1). They also show that nMA is higher in the suspension format independent of test conditions (Table S1).
HepG2. Induced ice nucleation was found to have a significantly positive effect on the nMA of HepG2 cells post-thaw in both formats (p-value < 0.001 for both monolayer and suspension; Figure 2). Model selection via AIC demonstrates that the most parsimonious model includes a fixed effect interaction term between ice nucleation and cell format, as well as a random intercept term for experimental replicate (Table S2). This linear mixed effect model is found to explain 86% (R squared = 0.86; Table   S2) of the variance in nMA. The model shows that both format and the variability associated with experimental replicate do have an effect on the relationship between ice nucleation and nMA. When ice nucleation is induced as opposed to passive, model estimates show that nMA is, on average, 61% and 8% higher in the monolayer and suspension formats, respectively (Table S1). They also show that nMA is higher in the suspension format (Table S1).

SW480.
Induced ice nucleation was found to have a significantly positive effect on the nMA of SW480 cells post-thaw in the monolayer format (p-value < 0.001; Figure 2). In comparison, no effect of induced ice nucleation is found in the suspension format (p-value = 0.38; Figure 2). Model selection via AIC demonstrates that the most parsimonious model includes a fixed effect interaction term between ice nucleation and cell format, as well as a random intercept term for experimental replicate (Table S2).
This linear mixed effect model is found to explain 88% (R squared = 0.88; Table S1) of the variance in nMA. The model shows that both format and the variability associated with experimental replicate do have an effect on the relationship between ice nucleation and nMA. Model estimates show that the nMA is, on average, 8% higher in the monolayer format when ice nucleation is induced compared to passive (Table S1). They also show that nMA is higher in the suspension format independent of test conditions (Table S1). Table S1. Fixed effect estimates and confidence intervals (lower = 2.5% -higher = 97.5%) for normalised metabolic activity (%) extracted from the most parsimonious models for each cell line (see Table 2). All values are rounded to two decimal places.

S12
Statistical analysis by cell linespheroids A549. Induced ice nucleation was found to have a significantly positive effect on the viability (V) of A549 cells post-thaw in the spheroid format (p value = < 0.001; Figure 3). This positive effect was found to occur in both sizes (4000 vs. 8000). We also find that a larger number of cells (8000) significantly increased viability. Model selection via AIC demonstrates that the most parsimonious model includes fixed effect terms for ice nucleation and size, however, an interactive term between the two explanatory variables is not supported (Table S3). This lack of support suggests that the expected positive effect of induced ice nucleation on viability is expected to be the same in either size. This linear mixed effect model is found to explain 56% (R squared = 0.56; Table 3) of the variance in viability.
Model estimates show that the viability is, on average, 29% higher under induced ice nucleation compared to passive ice nucleation (Table S4).
HepG2. Induced ice nucleation is found to have a significantly positive effect on the viability of cells post-thaw in the spheroid format (p value = < 0.001; Figure 3). This positive effect is found to occur in both sizes (4000 vs. 8000) but we find no significant effect of size on viability meaning that the positive effect of induced nucleation is independent of size. Model selection via AIC demonstrates that the most parsimonious model includes a single fixed effect term for ice nucleation (Table S3). This linear mixed effect model is found to explain 71% (R squared = 0.71; Table 3) of the variance in viability. Model estimates show that the viability is, on average, 49% higher under induced ice nucleation compared to passive ice nucleation (Table S5).  S13   Table S4. Fixed effect estimates and confidence intervals (lower = 2.5% -higher = 97.5%) for viability (%) of A549 cells extracted from the most parsimonious models (see Table 3). All values are rounded to two decimal places.  Table S5. Fixed effect estimates and confidence intervals (lower = 2.5% -higher = 97.5%) for viability (%) of HepG2 cells extracted from the most parsimonious models (see Table 3). All values are rounded to two decimal places.